Our research focuses on intrathymic T-cell development, which, as one of the few developmental programs that continue throughout life, is a privileged model to study cell death/survival decisions in mammals. After they have rearranged their T-cell Receptor (TCR) genes, immature thymocytes initiate the process known as T-cell selection, in which their fate, i.e. death vs. differentiation and survival, depends on signals generated by their TCR upon engagement by self-MHC peptide ligands. Our objective is to identify the intracellular signaling and gene expression programs involved in T-cell selection. Our first experimental approach has been to dissect this process into a sequence of genetically separable developmental steps by restricting the duration of intrathymic TCR signaling in vivo. To this end, we have generated a recombinant mouse model in which expression of Zap70, a tyrosine kinase required for TCR signal transduction, is confined to immature thymocytes. Using this model, we have shown that thymocytes subject to such developmentally confined TCR signaling initiate, but fail to complete, selection. Importantly, while they fail to develop into mature CD4+ or CD8+ T-cells, such "transiently" signaled thymocytes differentiate into the CD8 lineage, even if they harbor MHC II-restricted TCRs that normally are associated with CD4-lineage differentiation. These findings identify the duration of intrathymic TCR signaling as a critical determinant of CD4/CD8 lineage differentiation. By breeding these mice with mouse lines carrying defined genetic modifications, and by using high-throughput gene expression analyses, we are currently investigating the intracellular signaling pathways which transduce differentiation and survival signals during intrathymic selection. The other strategy we are pursuing to address these issues is to synchronize TCR signaling in vivo on thymocyte populations. This approach is of interest non only to T-cell development biology but also to the broader goals of identifying the mechanisms that maintain cell (or organism) homeostasis and of understanding how their disruption can lead to disease. Indeed, while "conventional" gene targeting or transgenesis are instrumental to address these issues, approaches with greater time resolution are needed to understand the dynamic of cell and organism responses to internal or external challenges. One such approach has been to target specific genes for time controlled expression, using ligand (e.g. tetracycline)-regulated expression systems; however, the temporal resolution of this method is limited by the unavoidable time lag required for de novo gene expression to achieve sufficient protein concentrations. To by-pass this limitation, our strategy has been to target protein function rather than gene expression, by developing proteins with ligand-controlled activity. We are using this strategy to achieve external control of TCR signal transduction by small diffusible ligands. We have validated this approach using transient expression in cell lines and we are planning to use it to study T-cell signal transduction and gene expression in vivo during intrathymic development.